Duchenne muscular dystrophy (DMD), an X-linked genetic disorder resulting from mutations in the dystrophin gene, afflicts 1 in 3300 males born each year in the US, causing devastating weakness and early death from cardiorespiratory failure. Most DMD patients develop pronounced cardiomyopathy by the second decade of life, with heart failure directly leading to death in about 30% of cases. However, little is known about the underlying cardiac pathophysiology in DMD patients. Golden retriever muscular dystrophy (GRMD) is the animal model that physiologically and clinically most closely resembles the human disease. Like humans with DMD, affected GRMD dogs display progressive heart failure leading to early death. Our long-term goal is to understand the cellular and molecular mechanisms by which dystrophin deficiency leads to cardiac abnormalities. In preliminary studies we have demonstrated markedly depressed contractility and impaired beta-adrenergic responsiveness in isolated cardiomyocytes from heart tissue of GRMD dogs compared with unaffected controls. However, the effects of general atrophy of skeletal muscles, including those supporting respiration, may indirectly contribute to the cardiac pathology in DMD. Therefore, it is imperative to develop an experimental system to study dystrophin deficient cardiomyocytes without the potential confounding effects of systemic disease. For this purpose we propose to utilize cardiomyocytes differentiated in vitro from pluripotent stem cells. The recent demonstration that it is possible to generate induced pluripotent stem (iPS) cells from easily obtained cells such as skin fibroblasts or keratinocytes, by relatively simple reprogramming steps, constitutes a major scientific advance with profound ramifications in many areas of basic research and applied clinical medicine. A powerful application of this technology is to obtain stem cell lines carrying medically significant gene mutations and to use these lines as a source of specialized cell lineages in order to assess effects of genotype on cellular phenotype in the absence of systemic perturbations. Our central hypothesis posits that loss of dystrophin in cardiomyocytes results directly in contractile dysfunction. To test this premise we will produce iPS cell lines from affected GRMD dogs and normal littermates, isolate cardiac lineage progenitors derived from these stem cells, and initiate assessment of the influence of the GRMD dystrophin mutation on cardiomyocyte development and function. Our Specific Aims are: Aim 1. To generate iPS cell lines from dystrophin-deficient (GRMD) dogs and normal controls, and confirm their character as pluripotent stem cells, in comparison with established canine embryonic stem (cES) cells. 1.1. Assess growth properties and pluripotency-associated markers, including cell surface antigens, transcription factors and telomerase. 1.2. Assess formation of teratoma tumors in immune deficient mice, and determine whether they contain cell types representative of the three embryonic germ layers. 1.3. Assess differentiation in vitro to cell types representative of all three germ layers. Aim 2. To examine the role of dystrophin in cardiomyocyte development. 2.1. Isolate cardiac progenitors from normal and dystrophin deficient canine iPS cell lines. 2.2. Isolate cardiomyocytes from normal and dystrophin deficient iPS-derived embryoid bodies. 2.3. Assess the expression levels of genes associated with cardiac development and maturation in cardiomyocyte lineage cells obtained in culture from normal and dystrophin-deficient iPS cells. Compare these with expression in cardiomyocytes from heart tissue of normal and GRMD dogs. 2.4. Compare the structure and organization of contractile proteins in developing normal and dystrophin- deficient cardiomyocytes generated in vitro from iPS cells. Also compare these with cells from cardiac tissue of normal and GRMD dogs. 2.5 Measure action potential recordings of canine iPS-derived canine cardiomyoctyes. The successful completion of these Aims will validate a unique GRMD model cell system that will complement in vivo studies and position us to fully characterize the direct effects of a dystrophin mutation on cardiomyocyte contractile function and other aspects of physiology. We anticipate that the iPS cell-based model will have great utility to study mechanisms of disease progression in skeletal and cardiac muscle, to screen for potential therapeutic agents, and to lay the groundwork for novel regenerative medicine approaches to treat DMD. PUBLIC HEALTH RELEVANCE: We hypothesize that the loss of dystrophin in cardiomyocytes causes a disease phenotype at the cellular level. We will test this premise in cardiomyocytes isolated from induced pluripotent stem cell lines derived from dogs harboring a dystrophin mutation.